While CRISPR-based genome engineering dominates the headlines these days, the natural function of CRISPR as a prokaryotic immune system is no less exciting. CRISPR is used by many bacteria and archea to fight off viral infections. It is a pretty effective tool and so phages had to evolve ways to suppress CRISPR immunity in order to be successful. Several phages encode proteins, so called anti-CRISPRs (Acr), which can bind to and neutralize CRISPR effector proteins in vitro. However, what role Acr proteins play in establishing phage infections in vivo is unclear. Phages infecting a CRISPR-expressing prokaryote enter a cell loaded with CRISPR effectors, which can destroy viral genomes within minutes. This raises the question whether Acr proteins, which are only expressed upon phage infection, can really act as effective immunosuppressants to protect the phage genome.

Two recent preprints now explore the role of Acr proteins during bacteriophage infections.

The Preprints / Key findings

The first preprint describes work by Mariann Landsberger and colleagues from Edze Westra and Stineke van Houte’s groups at the University of Exeter, while the second manuscript summarizes work by Adair Borges and others from Joseph Bondy-Denomy’s lab at UC San Francisco. Both groups started by analyzing the effect of different Acr proteins on phage infections of bacterial strains with and without CRISPR immunity. As expected, all phages successfully infected bacterial strains lacking a CRISPR immune system, while CRISPR cleared infections of phages lacking Acr genes. Interestingly, CRISPR-expressing bacteria were also protected from Acr-encoding phages added at low density and only succumbed to the infection when the viral load was high.

So why are Acr proteins an effective antidote to CRISPR at high phage density, but unable to protect phages at low density? One possibility would be that at high densities, bacteria are simultaneously infected by multiple phages and the higher load of viral genomes titrates away CRISPR effectors below a critical threshold. Borges et al. were able to exclude that hypothesis by showing that infections of an Acr phage are not helped by addition of a non-replicative phage strain that does not express an Acr proteins. Furthermore, the preprint by Landsberger and colleagues demonstrates that the critical density for successful infections by phages expressing a strong Acr protein occurs at conditions where simultaneous infections are rare. Instead, both groups propose a model where initial infections cause a immunosuppressed state that lasts long after the initial infection has been cleared. Phages that infect such immunosuppressed cells at a later stage can then successfully replicate. In support of this model, Borges et al. were able to show that transient infections of unsuccessful Acr phages can help subsequent infections to be successful. Furthermore, Landsberger et al. show that bacteria that had previously been infected with Arc phages are more susceptible to the transformation by plasmids for which they carry CRISPR spacers. In addition, both studies find that the phage density required for successful infections is inversely correlated with the affinity of the tested Arc protein for its CRISPR target protein.

Together, these data suggest that Acr proteins expressed by unsuccessful phage infections can induce an immunosuppressed state that can be exploited by subsequent phages infecting the same cell (see figure below from Borges et al.).

What I like about these pre-prints

The discovery and characterization of anti-CRISPR proteins has been one of the exciting developments in the CRISPR field in the past five years and has lead to a detailed biochemical and structural understanding of some Acr proteins in vitro. These two preprints now widen the focus and investigate the role of Acr proteins during phage infections. After all, as Dobzhansky famously remarked: “Nothing in biology makes sense except in the light of evolution”. With Dobzhansky’s words in mind, what I love about these preprints is that spinning the idea of an evolutionary arms race further readily gives rise to additional hypothesis that can be tested in the future (see below). If lingering Acr proteins cause immunosuppression, what mechanisms might have evolved in some prokaryotes to circumvent that? And if the currently known Acr proteins are unable to prevent elimination of the pioneer phage, what hitherto unknown mechanisms might have evolved in phages that don’t suffer this limitation? It seems certain that the golden age of CRISPR discoveries is far from over.

Besides the science, what I also like about these preprints is the fact that here two groups have investigated the same question and came up with the same answer and have decided to post their finding side-by side on a preprint server. Being afraid of being scooped ranks among the most cited reasons for scientists for being weary of posting preprints. In my view, these two preprints highlight two things: First, truly being scooped is extremely rare. That two teams independently investigating the same question would come up with an identical set of experiments is very unlikely. Rather than taking away from each other, these two manuscripts complement each other and together make a much stronger case for the conclusions than either manuscript alone would have done. Second, posting preprints gives authors control over the timepoint of when their manuscript will be publically available. This can be used to effectively coordinate the simultaneous release of complementary studies from different research teams. Maybe in the future preprint servers could offer additional tools to visibly link manuscripts reporting related findings.

Future directions

These preprints raise a number of questions that might be worth following up in the future.

Are Acr proteins on their own sufficient to cause lasting immunosuppression in bacteria? For example, would expression of a short pulse of Acr protein expressed from a heterologous plasmid be sufficient to allow subsequent phage infections at low density?

What is the importance of protein stability for the successful establishment of immunosuppression? Acr proteins that are more stable in the cytoplasm would be expected to provide a longer timeframe for secondary infections to occur. One prediction of that would be that prokaryotes might have evolved mechanisms to directly target Acr proteins for degradation.

What additional mechanisms might have evolveld in phages to circumvent elimination by CRISPR immune systems? Phages which rely on sequential infections of the same cell might have evolved ways to favor infections of immunosupressed cells. Furthermore, the relative inefficiency of Acr proteins in protecting the phage that encode them makes me wonder whether there might be other yet unidentified Acr proteins that are more potent and might be able to protect a phage that infects a cell with a fully functional CRISPR system. One might imagine enzymatic Acr proteins that can inactivate CRISPR proteins at sub-stoichiometric concentrations.